Field of the Invention
The invention relates to instruments and methods for the highly efficient acquisition of high numbers of substance-characteristic fragment ion species derived from precursor ions that have been separated by their mobility, in particular to quantify substances with accurate identity and improved signal-to-noise ratio.
Description of the Related Art
In protein science, there is an increasing interest in the quantification of peptides and proteins of a proteolytic digest of proteins extracted from a biological sample, performed by liquid-chromatography/mass-spectrometry (LC-MS). Special attention is directed also to the quantitative analysis of pharmaceutical drugs and their metabolites in pre-clinical studies. Furthermore, complex food/fruit/vegetable matrices may be tested for pesticides and other contaminants. Liquid chromatography coupled to triple quadrupole mass spectrometers renders low-price instrumentation known to be able to detect and to quantify substances with highest sensitivity, but the measurement methods are limited to a few substances appearing in an LC peak of limited duration. Unfortunately, increasing the number of substances by MRM (multiple reaction monitoring) decreases the measurement precision because lesser ions will be measured. In pre-clinical pharmaceutical studies, dozens of triple quadrupole instruments are operated in parallel to analyze hundreds and thousands of samples simultaneously.
U.S. Pat. No. 6,960,761 B2 (“Instrument for Separating Ions in Time as Functions of Preselected Ion Mobility and Ion Mass”, D. E. Clemmer, 2001) presents the application of one or more ion mobility separators in combination with mass filters and high-resolution mass analyzers. This document describes a large variety of methods to separate ions in mixtures using a number of different combinations of ion sources, ion traps, ion mobility separators, mass filters, collision cells, ion reactors, and high resolution mass analyzers. Furthermore, correspondent apparatuses are described as being composed of ion sources, ion traps, ion mobility separators, mass filters, collision cells, ion reactors, and high resolution mass analyzers in varying sequence. The high-resolution mass analyzer at the end of the method or device, either a time-of-flight (TOF) or an ion cyclotron resonance (ICR) mass spectrometer, measures the mass spectrum of the fragmented or unfragmented ions with high mass measurement precision. The time-of-flight mass analyzer (TOF) has the advantage of high speed measurements. The high-resolution mass analyzer, however, makes the whole instrument itself as well as its operation expensive. The relatively large physical size of the drift tube used by D. Clemmer in this device, orthogonal to the flight tube of the TOF-MS, makes it less feasible compared with the more compact footprint of modern mass spectrometers.
In U.S. Pat. No. 6,630,662 B1 (A. V. Loboda; 2002: “Setup for Mobility Separation of Ions Implementing an Ion Guide with an Axial Field and Counterflow of Gas”) an ion mobility separation device is described, mainly using a constant electric field and a profiled counterflow of gas. The device serves on one hand to collect ions from the ion pulses of a discontinuous MALDI ion source, and on the other hand, to separate the collected ions by their ion mobility. MALDI (matrix assisted laser desorption and ionization) mainly delivers singly charged molecular ions. Although the combination with a triple quadrupole mass spectrometer is briefly mentioned as a possibility, main attention is directed to a QqTOF, a combination of a mass filter (Q), a fragmentation cell (q), and time-of-flight mass analyzer (TOF) as the last mass analyzer, just as suggested by D. E. Clemmer.
A further ion mobility spectrometer has become known under the abbreviation “TIMS” (trapped ion mobility spectrometer). TIMS is a very small ion mobility spectrometer (the active part is only about five centimeters in length), quite different from both drift tubes at constant electric field and travelling wave mobility spectrometers. TIMS works with gas flow and electric counterfield and is thus more similar to the device of A. V. Loboda. In contrast to Loboda's device, an electric field profile with a ramped electric field barrier is used in a constant gas flow to hold back ions by their ion mobility; a decrease of the field barrier releases ions with increasing ion mobility, resulting in an ion mobility spectrum. The extraordinary and unique characteristic of ion mobility separators of the TIMS type is the fact that the ion mobility resolution continually increases with increasing scan duration. With TIMS, extraordinarily high ion mobility resolutions in the order of Rmob=400 have already been achieved experimentally. The TIMS is described in detail in U.S. Pat. No. 7,838,826 B1 (M. A. Park, 2008).
In co-pending U.S. patent application Ser. No. 14/931,163 (“Acquisition of Fragment Ion Mass Spectra of Ions Separated by their Mobility”; M. Mann et al., 2015), measuring methods for more than 300 fragment ion mass spectra per second with a TIMS-QqTOF are described with adjustable ion mobility resolution.
The instruments of the type QqTOF described so far all are high-priced instruments, a major share of the price being caused by the high-resolution mass analyzer. There is still a need for low-priced instruments and methods for the quantitative measurement of high numbers of characteristic fragment ions from proteolytic digests of proteins extracted from a biological sample, or from a complex food/fruit/vegetable matrix, to be tested for pesticides and other contaminants, without having to necessarily measure the whole fragment ion spectra.
Definitions
As usual in the mass spectrometric literature, the abbreviation “Q” stands for a quadrupole used as a mass filter and operated with radio frequency (RF) plus direct current (DC) voltages; whereas the abbreviation “q” concerns an RF quadrupole rod device essentially without DC voltages (sometimes called RF-only), used as ion guide or fragmentation cell. So the abbreviation “QqQ” stands for a triple quadrupole device with a fragmentation quadrupole between two mass filters, and “QqTOF” stands for a fragmentation quadrupole between a mass filter and a time-of-flight mass analyzer. Alternatively, the triple quadrupole instrument may be abbreviated Q1Q2Q3, particularly when the three RF quadrupole devices should be numbered, as is common practice in the art.
In some cases, the simplified expression “ion mass” stands here for the more exact “charge-related ion mass” (the ion mass m divided by the number z of surplus elementary charges of the ion), or the usual “mass-to-charge ratio m/z”.
The expressions “separator” and “separation” are used here for devices and methods which separate different substances or different ion species in time. Chromatography, capillary electrophoresis, and ion mobility spectrometry would fall under the definition of a separation method. The term “filter” is applied to devices and methods which are generally configured to let pass only selected ion species from a larger variety offered. An example is the RF quadrupole mass filter, capable of filtering ions by mass (though a skilled practitioner will understand that an RF quadrupole mass filter can usually also be switched into the RF-only mode having a comparatively broad bandpass characteristic).
The “mobility scan time” or “mobility scan duration” is defined as duration of an ion mobility scan over an interesting range of ion mobilities, usually the full range of the mobilities of the ions stored prior to the scanning.
The “ion mobility” K is defined as K=vd/E, vd being the drift velocity in a drift tube, E the electric field strength in the tube. A “reduced ion mobility” K0 is defined as ion mobility K at standard NTP conditions. The “ion mobility resolution” is defined as Rmob=K/ΔK, ΔK being the width of the ion mobility signal at half height.